tbuckley/cabot-search huggingface dataset:
TMIC-57. USING SINGLE-CELL SEQUENCING AND SPATIAL TRANSCRIPTOMICS TO IDENTIFY CANCER-ASSOCIATED FIBROBLASTS IN GLIOBLASTOMA AND DEFINING THEIR PRO-TUMORAL EFFECTS
Authors: Saket Jain, Jonathan Rick, Rushikesh Joshi, Angad Beniwal, Jordan Spatz, Sabraj Gill, Alexander Chang, Eric Chalif, Alexander F Haddad, Joseph F Costello, Aaron Diaz, Dieter Henrik Heiland, Manish Aghi
Journal: Neuro-Oncology (2022)

Abstract Cancer-associated fibroblasts (CAFs) constitute a key component of the tumor microenvironment. Pro-tumoral cancer-associated fibroblasts were presumed absent in glioblastoma given the lack of brain fibroblasts. Using single-cell RNA sequencing we identified CAFs in patient GBMs. CAFs were identified using a negative selection strategy to filter endothelial, epithelial, immune cells and pericytes and for the positive expression of previously defined CAF markers. Copy number variation (CNV) analysis was performed to distinguish CAFs from malignant cells. Single-cell spatial transcriptomics from 16 GBM patients confirmed the proximity of CAFs to mesenchymal GBM stem cells (GSCs), endothelial cells, and M2-macrophages. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and transcriptomic profile. CAFs were chemotactically attracted to GSCs and CAFs induced GSC proliferation. To identify CAF and GSC interaction mediators, we created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGFB as mediators of GSC effects on CAFs, and osteopontin and HGF as mediators of CAF-induced GSC enrichment. Furthermore, CAFs were found to induce M2-macrophage polarization by producing the EDA fibronectin variant which binds macrophage toll-like receptor 4 (TLR4) in a targetable manner. Glioblastoma CAFs were enriched in the subventricular zone which houses the neural stem cells that houses GSCs. Including CAFs in GSC-derived xenografts induced in vivo tumor growth and reduced survival in two different xenograft models. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target for Glioblastomas.
TMIC-54. THE ROLE OF TUMOR MICROENVIRONMENT DERIVED GROWTH FACTORS IN PEDIATRIC BRAIN TUMORS
Authors: Andrea Cruz, Abigail Locke, Katharine Halligan, Lauren Sanders, Allison Cheney, Ann-Catherine Jean Stanton, Robert Koncar, Alberto Broniscer, Olena M Vaske, Thomas Pearce, Daniel Marker, Clayton Wiley, Stephen C Mack, Mariella Filbin, Ian F Pollack, Sameer Agnihotri
Journal: Neuro-Oncology (2022)

Abstract BACKGROUND High-grade gliomas (HGGs) are the most common fatal intrinsic brain tumors in pediatric patients. H3K27-altered diffuse midline gliomas (H3K27-DMGs), a subgroup of HGGs defined by a histone 3 position 27 alteration, are especially aggressive and result in the poorest patient outcomes. Despite in-depth genomic characterization, the 5-year survival rate has yet to improve beyond 2% following diagnosis. A common feature of H3K27-DMGs is infiltration of microglia, macrophages, other myeloid cells, collectively referred to as GAMs, and a small population of T-cells. The contribution of non-tumor cells in the tumor microenvironment (TME) can both promote and or inhibit tumor growth, thus representing an opportunity in the pursuit of novel therapeutics. Using bioinformatic analysis on a human H3K37-DMG single cell-RNA sequencing dataset, we reveal several cell-to-cell communication signaling networks, mediated by ligand and receptor pairs, between GAMs and tumor cells, respectively. HYPOTHESIS Microglial-derived growth factors activate oncogenic signaling pathways via paracrine signaling axes, thus promoting H3K27-DMG tumor cell proliferation and growth. METHODS I will validate these findings and test their therapeutic potential using co-culture studies, CRISPR and shRNA gene silencing, and phospho-proteomics technology. RELEVANCE This research provides further insights on the contribution of non-tumor cells in the TME towards H3K27-DMG cell proliferation and growth and could potentially inform future therapy paradigms.
MODL-24. ESTABLISHING A CLINICALLY RELEVANT CT AND ASSOCIATED RADIOMICS PIPELINE FOR INTRACRANIAL RODENT TUMOUR MODELS
Authors: Kate Connor, Emer Conroy, Kieron White, Liam Shiels, Simon Keek, Abdalla Ibrahim, William Gallagher, James Clerkin, Kieron Sweeney, David O'Brien, Jane Cryan, Josephine Heffernan, Francesca Brett, Philippe Lambin, Henry Woodruff, Annette Byrne
Journal: Neuro-Oncology (2022)

Abstract While magnetic resonance imaging (MRI) is the predominant imaging modality for glioblastoma (GBM), pre-clinical MRI scanner availability is limited. As pre-clinical CT is more widely available and cost-effective, this study aimed to 1) establish preclinical-GBM CT and CT-radiomic workflows, 2) identify whether CE-CT could detect murine orthotopic GBM tumours on two CT instruments [TRIUMPHX-O-CT; IVISSPECTRUM-CT], 3) assess whether CT-radiomic features could distinguish tumour from normal tissue, and support earlier detection of tumours, 4) verify translation of pre-clinical CT-radiomic pipelines to, and assess pre-clinical CT-features in, clinical CE-CT scans.U87R-Luc2(n=25) and NFpp10a-Luc2(n=10) orthotopic GBM models were established and tumours monitored via bioluminescence imaging (BLI). Concurrently, mice underwent CE-CT (IV-iodine/300mg/mL/50kV-scan). Extracted radiomic features (PyRadiomics) underwent dimensionality reduction (Spearman correlation; &amp;gt;0.85). Remaining features were analysed (Recursive feature elimination (RFE)/RepeatedCV/randomforest) in normal and tumour tissue and across timepoints (TRIUMPHX-O-CT-Wk3vsWk6,Wk6vsWk9/12; IVISSPECTRUM-CT-Wk6vsWk9/12).CE-CT and radiomic pipelines were successfully established for orthotopic GBM models, using both CT-systems. On visual assessment of images, BLI was significantly more sensitive, with tumours detectable at Wk1 (BLI) vs Wk9 (CE-CT). However, RFE analysis identified CT-radiomic features (first_order&amp;glcm) which differentiated tumour from normal tissue (TRIUMPHX-O-CT). A subsequent feature set (first_order,glcm,gldm&amp;glzm) were identified (TRIUMPHX-O-CT/IVISSPECTRUM-CT), detecting tumours earlier (Wk3&amp;Wk6) than possible by visual assessment of CTs. Preclinical radiomic methods were successfully applied to exploratory clinical CE-CT scans(n=10). Here, several preclinical CT-features (e.g. Zone_Entropy) showed increased intensity in tumour regions. Overall experimental BLI is the most sensitive method for pre-clinical intracranial tumour detection. However, analysis of clinically relevant CT-radiomic features may facilitate tumour identification and earlier tumour detection (Wk3/Wk6-TRIUMPHX-O-CT/Wk6-IVISSPECTRUM-CT) than possible by visual assessment of CT (Wk9). Clinically relevant CT-derived radiomic features may therefore support intracranial rodent tumour assessment. Importantly, preclinical radiomic methods successfully translate to clinical CT-radiomic analysis. Parallel trends in tumour-specific feature intensities across pre-clinical and clinical scans suggest species conservation of features.
TMIC-33. ARGINASE-1 AND ITS ROLE IN THE GLIOBLASTOMA MICROENVIRONMENT
Authors: Daniel Zamler, Takashi Shingu, Shan Jiang, Fei Yu, Yingda Jiang, Giulio Draetta, Jian Hu
Journal: Neuro-Oncology (2022)

Abstract An average GBM has roughly 30% infiltration of myeloid cells, the highest reported case at 70% infiltration, with some variation dependent on subtype. When we consider this fact in combination with the advent and success of immunotherapies in similar cancer types, the logical next step follows that myeloid cells, which are of the immune lineage, have the potential to clear this aberrant growth. Known for their incredible phagocytic capacity, myeloid cells are among the first responders to an injury to control pathogens and clear apoptotic cells from the site of an insult. Myeloid cells, through poorly understood mechanisms, undergo a switch after 3-5 days from attacking pathogens and clearing dead cells to secretion of growth cytokines to promote wound healing. We posit these myeloid cells may be tricked into erroneously supporting the tumor cells in an attempt to “heal the wound” instead of mounting an immune response. The enzyme responsible for this switch from pathogen response to wound healing is called Arginase-1 (Arg1). When Arg1 is upregulated it has a two pronged effect, the first is creation of peroxynitrite which is actively immunosuppressive to both myeloid and T-cells. The second is an increase in ornithine when Arg1 cleaves arginine into ornithine and urea, which can be used to create the building blocks for cellular growth as well as to create collagen, which is necessary for wound repair and found in high levels in brain tumors. Our hypothesis is that through manipulation of the Arg1 axis in myeloid cells we will increase anti-tumor response. We show the groundwork for evaluating whether or not this is a viable therapeutic target in GBM as well as assess currently available compounds for inhibition or Arg1 in the CNS.
TMET-19. DEFINING A NOVEL ROLE FOR CD97 IN REGULATING GBM GLYCOLYTIC METABOLISM
Authors: Niklas Ravn-Boess, Nainita Bhowmick, Takamitsu Hattori, Varsha Prakash, Gabriele Stephan, Joshua Frenster, Devin Bready, Christopher Lawson, Jordan Wilcox, Dimitris Placantonakis
Journal: Neuro-Oncology (2022)

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Despite multimodal therapy, disease recurrence is inevitable. To identify novel vulnerabilities of GBM, we performed an arrayed CRISPR/Cas9 screen against select adhesion G protein-coupled receptors (aGPCRs), many of which we found to be de novo expressed in GBM. Knockout of CD97, previously implicated in GBM cell migration, produced the most striking proliferative disadvantage in patient-derived GBM cultures (PDGC) among aGPCRs tested. We found high CD97 surface expression in all our PDGCs, while levels remained nearly undetectable in non-neoplastic brain cells, confirming that CD97 is de novo expressed in GBM. Upon shRNA-mediated knockdown of CD97 in PDGCs from all three TCGA transcriptional subtypes, we observed reduced proliferation, as measured by cell cycle analysis. Notably, CD97 knockdown also significantly reduced tumorsphere formation capacity as measured by limiting dilution assays; an effect that was partially rescued upon CD97 overexpression. To elucidate mechanisms of action of CD97, we performed RNA-sequencing and GO pathway enrichment analysis from PDGCs following CD97 knockdown. The top downregulated pathways involved glycolytic metabolism, specifically involving many genes relevant for glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) processing. Indeed, when we measured metabolite levels under both steady-state and flux conditions using mass spectrometry, we observed an accumulation of G6P and a depletion of most downstream glycolytic and Krebs cycle metabolites upon CD97 knockdown. Furthermore, Seahorse metabolic assays revealed deficits in both glycolytic metabolism and oxygen consumption. We aim to interrogate the activity of specific glycolytic enzymes involved in processing G6P and F6P, pinpointing how these are influenced by CD97 signaling pathways (MAPK or Akt). Overall; our studies suggest a novel role of CD97 in regulating GBM metabolism (Warburg effect), and provide a strong scientific rationale for developing biologics to target CD97 which appears to be universally and de novo expressed in GBM.
TMIC-39. TARGETING EXTRACELLULAR MATRIX HYALURONIC ACID-CD44 SIGNALING REDUCES TUMOR STEMNESS AND SENSITIZES TUMOR TO VIROTHERAPY AND ENHANCES THERAPEUTIC POTENTIAL FOR CANCER TREATMENT
Authors: Upasana Sahu, Matthew P Mullarkey, Evan Hong, Pei Guangsheng, Zhao Zhongming, Bangxing Hong, Balveen Kaur
Journal: Neuro-Oncology (2022)

Abstract The tumor microenvironment (TME), including the non-tumor cells and the extracellular matrix (ECM) plays a crucial role in tumor progression and metastasis. Hyaluronic acid (HA), the major glycosaminoglycan present in brain ECM, has long been associated with the progression and invasiveness of brain tumors. HA signals primarily thorough CD44, an adhesion/homing receptor leading to the induction of cellular AKT, MEK and HIF signaling, thereby promoting tumor cell proliferation, aggressiveness and therapy resistance. While HA in the ECM has been shown to interfere with infection and spread of oncolytic viruses, the impact of tumor-ECM interaction induced signaling on oncolytic virotherapy is heavily understudied. RNA sequencing and gene set enrichment analysis of glioma cells infected with an oncolytic Herpes Simplex Virus-1 (oHSV) demonstrate an enrichment of pathways related to tumor-ECM interaction. Immunostaining of brain sections from intracranial tumor bearing mice also reveals increased HA after oHSV treatment. Our results further demonstrate that HA/CD44-mediated signaling inhibits virus replication in vitro. Herein, to evaluate the impact of blocking tumor-ECM interactions without altering the secreted ECM, we created oHSV-sCD44, an oHSV that encodes for extracellular soluble CD44 (sCD44) that can function as a dominant negative receptor for membrane bound CD44. oHSV-sCD44 significantly reduces the stemness of glioblastoma stem cells (GSCs), induces DNA damage and sensitizes the GSCs to radiation therapy. Intra-tumoral injection of oHSV-sCD44 into patient-derived primary GBM xenograft model significantly inhibits tumor growth accompanied by reduced stemness and decreased HA expression, and increased oHSV replication and tumor cell lysis in TME. Moreover, blocking HA-CD44 signaling with a single dose of oHSV-sCD44 in murine glioma syngeneic model induces the development of a significant anti-tumor immune response with enhanced T cell infiltration. Collectively, our findings implicate oHSV-sCD44 as a potential oncolytic and immune-stimulating anticancer therapeutic.
TMIC-41. ARGININE DEPRIVATION ALTERS DNA DAMAGE RESPONSE IN ARGINOSUCCINATE SYNTHETASE NEGATIVE GLIOBLASTOMA AND POTENTIATES STANDARD OF CARE
Authors: Avik Ghosh, Richard Perryman, Kevin O'Neill, Nelofer Syed
Journal: Neuro-Oncology (2022)

Abstract Glioblastoma (GBM) is the most aggressive and prevalent primary brain malignancy with an average life expectancy of 18 months despite the standard of care which includes resection, temozolomide and radiotherapy. Thirty percent of GBMs show transcriptional loss of arginosuccinate synthetase 1 (ASS1), displaying arginine auxotrophy and hence, are amenable to arginine deprivation therapy by ADI-PEG20. Moreover, these tumours are more aggressive than their non-arginine auxotrophic counterparts. In this study we demonstrate that this may lie in part due to their enhanced ability to repair damaged DNA. ADI-PEG20 significantly altered the expression of DNA repair genes in vitro and in vivo. We observed downregulation of PCNA, ATR (p=0.0063) and CHEK1 (p=0.0150) and upregulation of CHEK2 (p=0.0033) and RAD51 (p=0.0230). PARP1 was also downregulated, albeit not statistically significant. Addition of ADI-PEG20 to temozolomide and radiation also caused significant downregulation of BRCA1 (p=0.0340) . With DNA repair gene upregulation contributing to treatment resistance and DDR inhibitors shown to potentiate current therapies in the literature, not only do our results help elucidate the mechanisms of radiosensitisation with ADI-PEG20, targeting this pathway with DDR inhibitors alongside the pleiotropic effects of ADI-PEG20 may provide a novel therapeutic combination for ASS1 negative GBM.
TMIC-65. DISSECTING THE EXPRESSION AND FUNCTION OF ANGIOPOIETIN-1 IN BRAIN TUMORS
Authors: Jaldeep Langhnoja, Xin Wei, Phonepasong Arounleut, Timothy Phoenix
Journal: Neuro-Oncology (2022)

Abstract Angiopoietin-1 (Angpt1) is a secreted protein that can promote angiogenesis and vascular stability in development and certain disease states through activation of the Tie2 receptor. However, little is known about its expression or function across brain tumor types. Surveying its expression across pediatric and adult brain tumors we show Angpt1 is upregulated in glial brain tumors, including high-grade gliomas and ependymomas, and that expression patterns correlate with cell-type expression patters we recently described in the normal developing and adult brain. To examine the cell type and location of Angpt1 expression within tumors we have generated electroporation based high-grade glioma and ependymoma mouse models in Angpt1-GFP reporter knock-in mice. Angpt1-GFP expression is found mainly in tumor cells within both tumor models, with varying expression in high-grade glioma models based on driver mutations and molecular subtype. Single-cell transcriptomic studies in these mouse models are underway to further delineate expression across tumor and non-tumor cell types. Moreover, we are leveraging this flexible electroporation-based mouse brain tumor modeling platform to examine Angpt1 function within brain tumors. Preliminary data shows that including a DNA plasmid encoding Cre-recombinase induces recombination and loss of Angpt1 in our electroporation-based brain tumor models, and ongoing studies aim to delineate its function in tumor pathogenesis, including tumor angiogenesis and blood-brain barrier function.
TMIC-28. COMBINING INDUCED NEURAL STEM CELL THERAPY AND IMMUNOMODULATION IN GLIOBLASTOMA
Authors: Morrent Thang, Alain Valdivia, Shawn Hingtgen
Journal: Neuro-Oncology (2022)

Abstract Glioblastoma (GBM) is an aggressive malignant adult brain tumor and is characterized by poor prognosis and survival outcome. Insufficient drug accumulation to the tumor site and development of monotherapy drug resistance are critical challenges to overcome in combating GBM. Human induced neural stem cells (hiNSC) therapy is promising due to the stem cells’ innate capability to migrate to the tumors and deliver therapeutics. We previously have developed spheroidal iNSCs and engineered them to constitutively secrete pro-apoptotic protein TRAIL—hiNeuroS-TRAIL. However, investigating a combination therapy along with hiNeuroS-TRAIL therapy is needed to potentially avoid monotherapy resistance and enhance overall treatment durability. One of the GBM hallmarks is its immunosuppressed tumor microenvironment (TME). GBM-associated macrophages and microglia (GAMs) display pro-tumoral phenotypes in TME—inhibiting their ability to target the tumor cells or recruit other immune cells. In humans, higher glioma grade is associated with higher expression of Mer Tyrosine Kinase Receptor (MerTK) in both GBM cells and GAMs. Activation of MerTK in GAMs and GBM cells exacerbates immunosuppression and tumor progression respectively. Inhibiting MerTK activation via a small molecule drug, MRX-2843 and combining with hiNeuroS-TRAIL demonstrated robust synergistic tumor killing effects in vitro (CDI=0.22) and ex vivo (CDI=0.18). Additionally, the preliminary data for in vivo pilot study revealed that the median survival for the combination therapy treated group of mice extended over 24 days whereas the median survival for control, MRX-2843, and hiNeuroS-TRAIL treated groups of mice were 16, 18, and 21 days respectively. We think that combining these two therapies could significantly extend the survival in our murine model of GBM. Therefore, we will be optimizing dose concentration and cell counts for single agent therapies, and we will then repeat in vivo y synergy study with optimized therapies and a larger sample size to fully uncover the potential of this combination therapy.
MODL-23. MULTISCALE MODELS OF GLIOMA PROGRESSION: INSIGHTS ABOUT VASCULATURE AND THERAPY EFFECTS
Authors: Martina Conte, Christina Surulescu, Yvonne Dzierma, Sven Knobe
Journal: Neuro-Oncology (2022)

Abstract Gliomas are the most prevalent, aggressive, and invasive subtype of primary brain tumors, characterized by fast cell growth, strong invasion capability, and well-developed tumor vasculature. Although the research advances and the clinical trials testing the efficacy of novel combined therapies have allowed significant progress in the comprehension and treatment of gliomas, these tumors are characterized by a poor prognosis, and recurrence remains the main cause of mortality. The growth and migration of tumor cells inside the brain is a highly complex phenomenon, influenced by a multitude of intrinsic and extrinsic factors at different spatial and temporal scales. The high complexity of this invasion process remains a challenge to face and several important questions are still unanswered. In this context, mathematical models emerge as powerful tools to face these challenges. Here, we propose a multiscale approach for the description of glioma progression under the influence of vascularization. Precisely, we firstly focus on the effects of (hypoxia-driven) acidity and phenotypic heterogeneity on tumor progression. Then, we extend the setting to include vascular endothelial growth factor evolution and compare various therapeutic approaches. Starting from a microscopic description of the interactions between specific cell membrane receptors and the extracellular microenvironment, we define two different coupled systems for endothelial and tumor cell evolution. The analysis of these systems allows us to assess tumor growth and spread on the basis of DTI and glioma patient data. In particular, we perform several data-driven numerical tests aimed at assessing the role of vasculature and phenotypic heterogeneity on tumor progression and comparing the effect of different combinations of radio-, chemo-, and anti-angiogenic therapy on it.
TMIC-12. COMPLEMENT SIGNALING IN HYPOXIC NICHES OF THE GLIOMA MICROENVIRONMENT
Authors: Rebecca Rosberg, Karolina Smolag, Jonas Sjölund, Vasiliki Pantazopoulou, Kristian Pietras, Anna Blom, Alexander Pietras
Journal: Neuro-Oncology (2022)

Abstract Glioblastoma is the most common and aggressive primary brain tumor in adults. Despite treatment through surgery, irradiation and chemotherapy, all patients suffer recurrence of treatment-resistant tumors. Previous studies from our lab showed that astrocytes become reactive and generate tumor-supportive conditions for glioma cells, when exposed to hypoxia or irradiation. Reactive astrocytes are known to upregulate proteins of the complement system, especially complement component 3 (C3), in several neurological disorders. However, it remains unexplored how these complement proteins are expressed in stromal astrocytes in glioblastoma. Tissue sections from a glioma mouse model showed presence of C3 in the invasive front, and in hypoxic, and perivascular spaces of the tumors, where there is an abundance of astrocytes. C3 expression co-localized with Nestin and CD44, two markers of mesenchymal and/or stem-like glioma cells. Primary human astrocytes grown in hypoxic conditions upregulated C3 as well as other proteins associated with a more extensive infiltrative phenotype of glioblastoma. In general, a strong correlation between hypoxia (195 genes) and complement (200 genes) gene signatures were found in TCGA GBM dataset (R= 0.82, p-value= 0). Single-cell sequencing data from primary GBM tumors showed a subpopulation of highly C3-expressing astrocytes, which were enriched for cellular pathways comprising epithelial mesenchymal transition (EMT), TNF-alpha, complement, hypoxia, and interferon signaling. Human glioma cell lines from proneural, classical and mesenchymal subtypes all showed increased CAIX and GLUT1 expression grown under lower oxygen tensions, while two out of three subtypes showed upregulation of stemness markers (OCT4 and NANOG1) as well as C3 and C3aR. Overall, our data indicate a strong link between hypoxia and complement expression in the brain tumor microenvironment, where a local expression of complement proteins could possibly lead to tumor promoting signaling, which will be further explored.